Differentiation of Classical Swine Fever Virus Virulent and Vaccine Strains by CRISPR/Cas13a

ABSTRACT As a notifiable terrestrial and aquatic animal disease listed by World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]), classical swine fever (CSF) has caused great economic losses to the swine industry worldwide during recent decades. Differentiation of infected and vaccinated animals (DIVA) is urgent for eradication of CSF. In this study, a diagnostic platform based on CRISPR/Cas13a was established with the ability to differentiate between classical swine fever virus (CSFV) virulent and vaccine strains. In combination with reverse transcription recombinase-aided amplification (RT-RAA), the detection limit for CSFV synthetic RNA templates reached 3.0 × 102 copies/μL. In addition, with boiling and chemical reduction, heating unextracted diagnostic samples to obliterate nucleases (HUDSON) treatment was introduced to inactivate nucleases and release viral genome, achieving robust pretreatment of tested sample before CRISPR/Cas13a detection without the need to extract viral nucleic acids. HUDSON-RT-RAA-CRISPR/Cas13a can directly detect cell cultures of virulent Shimen strain and vaccine hog cholera lapinized virus (HCLV) strain, with the detection limit of 3.5 × 102 copies/μL and 1.8 × 102 copies/μL, respectively, which was equally sensitive to nested PCR (nPCR) and 100 times more sensitive than antigen enzyme-linked immunosorbent assay (ELISA). Meanwhile, HUDSON-RT-RAA-CRISPR/Cas13a showed no cross-reactivity with bovine viral diarrhea virus (BVDV), atypical porcine pestivirus (APPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), pseudorabies virus (PRV), and porcine circovirus 2 (PCV2), exhibiting good specificity. At last, a total of 50 pig spleen samples with suspected clinical signs were also assayed with HUDSON-RT-RAA-CRISPR/Cas13a, nPCR, and antigen ELISA in parallel. HUDSON-RT-RAA-CRISPR/Cas13a showed 100.0% with nPCR and 82.0% coincident rate with antigen ELISA, respectively. IMPORTANCE Classical swine fever (CSF) is a World Organisation for Animal Health (formerly the Office International des Epizooties [OIE]) notifiable terrestrial and aquatic animal disease, causing great economic losses to the swine industry worldwide during the past decades. Due to the use of the most effective and safe attenuated live vaccine for CSF prevention, differentiation of infected and vaccinated pigs is vital work, as well as a bottleneck for eradication of CSF. Methods with the ability to precisely differentiate classical swine fever virus (CSFV) virulent strains from vaccine strain hog cholera lapinized virus (HCLV) are urgently needed. Combining the high sensitivity of isothermal recombinase-aided amplification (RAA) with the accurate molecular sensing ability of Cas13a, we presented a novel method for CSFV detection without the need to extract viral nucleic acids, which showed great advantage to traditional detection methods for precise differentiation of CSFV virulent strains and vaccine strain, providing a novel powerful tool for CSF eradication.

In this study, the authors established a method for the differentiation of Classical swine fever virus virulent and vaccine strains by using HUDSON-RT-RAA-CRISPR/Cas13a, the sensitivity and specificity were also evaluated. How, the present assay established need be further evaluated. As a CRISPR detection method, the sensitivity in this assay just only reaches 300 copies/μL. At present, I have checked many publications on CRISPR detection methods, and the detection limit can be reached 10 copies or even 1 copy per μL. Additionally, the comparative experiment with ordinary PCR method is done in this assay, and the sensitivity is the same each other, which causes that this assay has no practical significance in the field, and the sensitivity is not up to that of qPCR. Therefore, I suggest further optimization of the method. Other points as follow: 1.In Figure 1, the detection method is divided into 3 steps. It is whether the reagent is added by opening the lid according to the steps? If it is added in steps, it is concerned about the possible nucleic acid aerosol contamination after opening the lid between the second and third steps. 2.In Figure 2, the annotation of marker does not match the figure . 3.In Figure 3, the representation of the 3A and 3B is reversed. 4.In Figure 9, many non-specific bands can be seen, and I have doubts about the results of this PCR. 5.It is recommended to incorporate some figures and tables to enhance the readability of the manuscript. 6.Line 309,cNDA should be modified to cDNA；Line 311 ,PCV 2 should remove the space.
Reviewer #3 (Comments for the Author): n the present study, Zhang et al. reports a CRISPR/Cas13a based diagnostic method for differential detection of virulent and vaccine strains of classical swine fever virus (CSFV). Considering CSF eradication is undergoing in China and countries that use hog cholera lapinized virus (HCLV) derived live attenuated live vaccines, this method has important research significance for it provides a promising strategy for CSF diagnosis and eradication. However, there are still some major and minor concerns that need author to be improved as following: Major concerns: 1. Line 33, " In combination with recombinase-aided amplification (RAA) ". As it is known, there are currently two recombinase based amplification techniques, including recombinase polymerase amplification (RPA) and recombinase-aided amplification (RAA), of which RPA is the most popular amplification technique that combined with CRISPR. What are the differences between RPA and RAA? Is there any reason that RAA is chose in the present manuscript? 2. Line 115, " 50 spleen tissues collected from pigs with suspected clinical signs". This clinical signs should be described more specifically. In other words, what signs that are likely to be caused by CSFV and may confused with other porcine virus infection. 3. Line 163, " Viral cell cultures or tissue honogenates (100 mg sample in 500 μL PBS)". It is confused to understand how tissue honogenates were prepared, a more detailed description is needed to be clarified. 4. Line 219, figure 8 and figure 11, " results were calculated according to the manufacturer's instruction". There is a need to introduce how to decide the ELISA results. Similarly, how to determine the positive and negative lines in figure 8 and figure 11? Minor concerns: 1. There are some abbreviations such as CSFV, HUDSON, etc., should be given the full name when they appeared for the first time. 2. Line 110, "synthesized by Sangon Biotech". Please indicate the country of the company. In addition, all regents and company in this manuscript should also indicate country.
Staff Comments:

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Revision Notes
Dear Editor: Thank you for your kind letter of "Spectrum00891-22 Decision Letter" on April 4th, 2022.
Generally, we appreciate the editor and reviewer's insightful comments, which were helpful for improving the manuscript. Based on your comments and requests, we have made modification on the original manuscript.
Here are some issues need to communicate with editor: 1. The name of author's department 1 was more specifically changed from "College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, China" to "International Joint Research Center of National Animal Immunology, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, China".
2. Bo Wan, the vice professor from Henan Agricultural University, has made great contribution during processing the revised manuscript, including providing necessary experimental materials and improving manuscript's quality. Thus, we decided to listed Bo Wan as an additional co-author.
3. There is a mistake of second co-author. During the first vision of the manuscript, the name of second co-author is "Qimei Li" on the website of the journal, while the correct name is "Qingmei Li". We failed to correct the name online, and we are hopeful to ask editor's kind help to correct the name. 4. As listed in the manuscript. there are two corresponding authors in the manuscript. The first corresponding author is Gaiping Zhang, while the second one is Junqing Guo. However, we cannot choose Junqing Guo as corresponding author online. We'd like to know if Microbiology Spectrum only allows one corresponding author? If not, we are hopeful to ask editor's kind help to choose Junqing Guo as second corresponding author.
Thanks again for insightful comments from editor and reviewers. We have modified the manuscript and made point-to-point response according to reviewers' comments. In revision notes, the line numbers refer to the PDF vision of the revised manuscript.

Responses to reviewer #1 comments:
We have highlighted the changes in yellow according to reviewer #1 comments in the revised manuscript.
The present study by Yuhang Zhang et al. developed a CRISPR/Cas13a-based novel method for differential detection of wild-type classical swine fever virus and C-strain. The method was evaluated to be sensitive and specific. However, several concerns need to be addressed.
Many thanks. We appreciate the reviewer's carefulness and insightful comments. Some details have been clarified as follows.

More reference CSFV strains of different genotypes and more clinical samples of different types should be included to evaluate the established method.
Re：We are thankful to the reviewer's insightful comments. Currently, there are three genotypes and eleven subgenotypes based on partial sequences of 5'UTR, E2 and NS5B. However, limited information of 3'UTR is available on NCBI and published studies. In the present manuscript, we systematically estimated the ability of established CRISPR/Cas13a to recognize all CSFV genotypes whose sequential information can be found, including subgenotype 1. During development of CRISPR/Cas13a, we actually tested different organs of two infected pigs, including heart, liver, spleen, lung, kidney, brain and lymphatic node. Our results showed that spleen and lymphatic node from both pigs showed the highest fluorescent signals. In addition, in comparison experiment of clinical samples between CRISPR/Cas13a, nPCR and antigen ELISA, we further considered spleen was one major virus carrier for most porcine diseases, which means spleen was most likely to be infected with different viruses, thus we obtained fifty spleen samples to estimate the performance of established CRISPR/Cas13a assay.

Other pestiviruses, including atypical porcine pestivirus and border disease virus, need to
be included in the specificity test.
Re：We are thankful to the reviewer's insightful comments. In the previous manuscript, only one pestivirus, bovine viral diarrhoea virus (BVDV), was assayed in the specificity test, which might not convinsible to declare the specificity of the established CRISPR/Cas13a method. Thus, we prepared cDNA from one porcine tissue infected by atypical porcine pestivirus (APPV), verified it by PCR and then tested it with CRISPR/Cas13a. Results showed the prepared cDNA was verified as APPV-positive by PCR, while CRISPR/Cas13a showed no cross-reactive with this sample.
As another member of pestiviruses, even though border disease virus (BDV) is less harmful to pigs compared with APPV and BVDV, it is also considered to be tested by CRISPR/Cas13a. We keep contacting with different colleges and organizations to search for a BDV positive sample once we received comments of reviewer #1. Unfortunately, we failed to obtain one at present.
Cas13a is a CRISPR effector with high specificity for molecular sensing, only tolerating no more than two mismatches. We aligned our crRNAs with the genome of BDV published on NCBI

The manuscript should be revised by native English speakers.
Re: We are thankful to the reviewer's insightful comments. We have asked for native English speakers and colleagues who once lived in English-speaking countries to revise throughout this manuscript. Detailed modifications are listed below: Corrected mis-spellings: Line 162: "patial" was changed to "partial".

Corrected grammatical mistakes:
Line 255-256: "followed by boiling for 5 min to lysis viral particles and inactivate nucleases" was changed to "followed by boiling for 5 min to lyse viral particles and inactivate nucleases".
Line 234: "as well as ASFV OIE recommended PCR were lists in Table 4" was changed to "as well as ASFV OIE recommended PCR were listed in Table 4".
Line 338-339: "which were coincident with the detection limits when testing synthetic RNA templates" was changed to "which were coincident with the detection limits for testing synthetic RNA templates". Line 346-347: "Results showed a complex infection situations among these samples" was changed to "Results showed the infection situations were complex among these samples".

Reviewer #2 (Comments for the Author):
In this study, the authors established a method for the differentiation of Classical swine fever virus virulent and vaccine strains by using HUDSON-RT-RAA-CRISPR/Cas13a, the sensitivity and specificity were also evaluated. However, the present assay established need be further evaluated. As a CRISPR detection method, the sensitivity in this assay just only reaches 300 copies/μL. At present, I have checked many publications on CRISPR detection methods, and the detection limit can be reached 10 copies or even 1 copy per μL.
Additionally, the comparative experiment with ordinary PCR method is done in this assay, and the sensitivity is the same each other, which causes that this assay has no practical significance in the field, and the sensitivity is not up to that of qPCR. Therefore, I suggest further optimization of the method.
Re: Many thanks. We appreciate the reviewer's carefulness and insightful comments. We have highlighted changed contents in green.
As is mentioned in the manuscript, the main disadvantage of CRISPR is poor detecting sensitivity. Thus, pre-amplification of tested samples by PCR and isothermal amplification techniques is the main strategy for sensitivity enhancement. Currently, there is no fixed rules to estimate the performance of primers used for RPA or RAA. Development of RAA with high sensitivity solely rely on large-scale work for candidate primers screening. Considering the main purpose of our work is to differentiate CSFV virulent and vaccine strains, our detecting target focused on the area of 12 nt-insertion at 3'UTR, which limits the number of candidate primers to optimize. Once received comments of Reviewer #2, we screened candidate primers which meet the principles of RAA primer designing around 12 nt-insertion and all candidate primer pairs showed similar performance compared with initial primer pairs. Added contents have been presented in supplementary materials as figure S1 and table S1. Other points as follow: 1. In Figure 1 Properly dispose tips, strips, excess buffer and other related materials after RAA experiments. Figure 2, the annotation of marker does not match the figure.

In
Re: We are thankful to the reviewer's insightful comments. The annotation of marker has been modified to match the figure. Figure 3, the representation of the 3A and 3B is reversed.

Re:
We are thankful to the reviewer's insightful comments. The presentation of figure 3 has been corrected.
Changed content in line 568-575: The figure captions were modified to match figure 3. Figure 9, many non-specific bands can be seen, and I have doubts about the results of this PCR.

Re:
We are thankful to the reviewer's insightful comments. In figure 9, nPCR for PEDV of some

Re:
We are thankful to the reviewer's insightful comments. We have merged figure figure 9,  However, there are still some major and minor concerns that need author to be improved as following: Re: Many thanks. We have exhibited changed contents with red color according to reviewer #3 comments in the revised manuscript. Re: We are thankful to the reviewer's insightful comments. Currently, there are two kinds of recombinase-based amplification techniques, which are recombinase polymerase amplification (RPA) and recombinase-aided amplification (RAA). In general, RPA and RAA share the same steps and mechanism for nucleic acid amplification. The only difference between RPA and RAA is the source of recombinase used. The recombinase used in RPA reaction is from T4 phage, while the recombinase used in RAA reaction is from E.coli.
Though RPA is the first amplification technique combined with CRISPR technique, there are increasing number of studies using RAA to improve detecting sensitivity as well. More importantly, in a recent paper about rapid detection for African swine fever virus (ASFV), RPA and RAA were compared in parallel. Results showed that RAA was about two-fold more sensitive than RPA for ASFV detection in that paper. Thus, we finally chose RAA in our study.

Revision Notes on manuscript "Spectrum00891-22R1"
Dear Editor: Thank you for your kind letter of "Spectrum00891-22R1" on June 18, 2022. Generally, we appreciate the editor's insightful comments, which were helpful for improving the manuscript. Based on your comments and requests, which is "merging some related figures into one, or putting over-sized figure(s)/table(s) into supplemental information", we have made modifications in the manuscript Spectrum00891-22R1 as following and highlighted changes in blue in the revised one: